Blocking Western with Fab fragment - (Jun/20/2006 )

Hi there,

have do put a secondary anti-mouse on my mouse tissue blot.

Of course get many unspecific bands presumably due to internal IgG in my tissue.

In Immunohistochemistry there is the possibility to block these by pre incubation with unconjugated anti-mouse fab fragment (http://www.ihcworld.com/_protocols/general_IHC/immuno_mom.htm).

Anyone ever tried that on Westerns?

Seems impossible to just lower the anti-mouse concentration without loosing the wanted signal of my monoclonal primary ab to....

no experiences or suggestions?

anything???

Interesting.
I never tried to block with Fab, but I don't see any reason why it would not work.

As I do IP with murine Ig, and detect also with a murine Ig, I don't use an anti-mouse antibody, but I prefer to use protein A or protein G coupled to HRP. It works quite fine.

it works:

background signal is getting much weaker (see attachment)

at the moment trying with primary antibody included: wish me luck